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WO2015058111A1 - Nanoparticules cationiques pour co-administration d'acides nucléiques et d'agents thérapeutiques - Google Patents

Nanoparticules cationiques pour co-administration d'acides nucléiques et d'agents thérapeutiques Download PDF

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Publication number
WO2015058111A1
WO2015058111A1 PCT/US2014/061185 US2014061185W WO2015058111A1 WO 2015058111 A1 WO2015058111 A1 WO 2015058111A1 US 2014061185 W US2014061185 W US 2014061185W WO 2015058111 A1 WO2015058111 A1 WO 2015058111A1
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WIPO (PCT)
Prior art keywords
sirna
nanoparticle
compound
cells
hydrophobic
Prior art date
Application number
PCT/US2014/061185
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English (en)
Inventor
Xiaoyang Xu
Xueqing Zhang
Omid C. Farokhzad
Robert S. Langer
Original Assignee
The Brigham And Women's Hospital, Inc.
Massachusetts Institute Of Technology
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Filing date
Publication date
Application filed by The Brigham And Women's Hospital, Inc., Massachusetts Institute Of Technology filed Critical The Brigham And Women's Hospital, Inc.
Priority to US15/029,084 priority Critical patent/US10874621B2/en
Publication of WO2015058111A1 publication Critical patent/WO2015058111A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes

Definitions

  • This invention is in the field of nanoparticles, particularly nanoparticles which can effectively co-deliver nucleic acids, such as siRNAs, and small molecule therapeutics.
  • cancer is usually the result of a combination of interconnected disease pathways that may not be treated effectively with one-dimensional therapeutic
  • abiraterone or enzalutamide results in the development of abiraterone or enzalutamide refractory castration resistant prostate cancer (CRPC) that is phenotypically non-adenocarcinoma and represents a rare and often lethal form of prostate cancer with neuroendocrine phenotype.
  • CRPC castration resistant prostate cancer
  • Cisplatin and other DNA adduct-forming chemotherapeutics cause DNA damage as their primary mechanism of cellular cytotoxicity.
  • DNA repair pathways that remove the damage
  • human translesion synthesis (TLS) by specialized DNA polymerases that help the cells tolerate the DNA damage.
  • Revl/Rev3L/Rev7-dependent error-prone TLS pathway has been shown to play an important role in cisplatin-induced mutations that improve the capacity of tumor cells to either repair or tolerate DNA damage, resulting in acquired chemoresistance.
  • Recent studies using mouse lymphoma and lung cancer models have shown that the suppression of crucial gene products (Revl and Rev3L) involved in the error-prone TLS activity in mammalian cells can inhibit drug-induced mutagenesis so that relapsed tumors remain sensitive to subsequent treatment. It has been suggested that combining conventional chemotherapy with newly emerging siRNA therapeutics could be a promising strategy for improving the efficacy of chemotherapy through additive or synergistic effects.
  • RNA interference RNA interference
  • synthetic siRNA Since the discovery of RNA interference (RNAi), synthetic siRNA has emerged as a class of attractive therapeutics for treatment of various diseases including cancer. Given the ability to target and silence nearly any gene of interest, specific siRNA can be constructed to target genes encoding proteins involved in DNA repair and the acquisition of multidrug resistance (MDR). Naked siRNA cannot readily cross cellular membranes due to its polyanionic and macromolecular characteristics, and it is susceptible to degradation by endogenous enzymes. Therefore, considerable efforts have been made to develop safe and effective vehicles in order to facilitate the delivery of siRNA into cells. Similarly, the methods by which RNA interference (RNAi), synthetic siRNA has emerged as a class of attractive therapeutics for treatment of various diseases including cancer. Given the ability to target and silence nearly any gene of interest, specific siRNA can be constructed to target genes encoding proteins involved in DNA repair and the acquisition of multidrug resistance (MDR). Naked siRNA cannot readily cross cellular membranes due to its polyanionic and macromolecular characteristics, and it is susceptible to degradation
  • chemotherapeutics are delivered also have a significant effect on the efficacy. Recent research has begun to explore the feasibility of combining chemotherapeutics with siRNA using a variety of nanocarrier platforms.
  • nanocarriers such as nanoparticles, which effectively co-deliver a combination of nucleic acids, such as siRNA, and small-molecule therapeutics, such as chemotherapeutic agents, and methods of making and using thereof.
  • Nanoparticles containing an aqueous core containing one or more nucleic acids, such as siRNA, and a shell containing one or more hydrophobic cationic materials, one or more amphiphilic materials, and one or more therapeutic, diagnostic, and/or prophylactic agents have been developed.
  • the hydrophobic cationic material and the hydrophobic portion of the amphiphilic material provide a non-polar polymer matrix for loading non-polar drugs, protect and promoting siRNA molecule retention inside the NP core, and control drug release.
  • the hydrophilic portion of the amphiphilic material can form a corona around the particle which prolongs circulation of the particles in the blood stream and decreases uptake by the RES.
  • Preferred siRNA molecules include those that inhibit the DNA repair pathways and human translesion synthesis (TLS) by specialized DNA polymerases that help the cells tolerate the DNA damage such as the Re l/Rev3L/Rev7-dependent error-prone TLS pathway. These are preferably administered in combination with chemotherapeutic compounds such as ciplatin or other platinum derivatives.
  • TLS human translesion synthesis
  • Figure 1A is a schematic of PLGA-WEG/G0-C14 NPs.
  • the particle contains three components: i) an outer PEG surface; ii) a PLGA/G0-C14 layer which plays two roles: a) carrying non-polar drugs in a polymer matrix and b) protecting and promoting siRNA molecule retention inside the NP core and controlling drug release; and iii) an aqueous inner core containing siRNA.
  • Figure IB is the chemical structure of the hydrophobic platinum (IV) compound 1 and the chemistry by which the active drug, cisplatin, is released, after reduction in the cell.
  • Figure 1C is the scheme for the synthesis of G0-C14 through ring-opening of 1,2-epoxytetradecane by
  • Figure ID is a graph showing the size distribution of the NPs containing both compound 1 and siRNA determined by dynamic light scattering (DLS).
  • Figure 2 is a graph showing the in vitro release profile of the siRNA and compound 1 from PLGA-0-PEG/G0-C14 NPs (percent drug release) as a function of time (hours).
  • Figure 3A is a graph showing the relative firefly luciferase expression of Dual-Luc HeLa cells transfected with NP(siLuc) at escalating dose of siLuc. Relative firefly luciferase expression was determined by comparison of detected protein levels in treated groups vs. untreated control. Lipo2000- siRNA complex containing 50 ng siRNA was used as a positive control.
  • Figure 3B is a graph showing the viability of Dual-Luc HeLa cells incubated with escalating dose of NP(siLuc).
  • Figure 4 is a graph showing in vivo bioluminescence imaging.
  • Luciferase-expressing xenograft tumors were induced in the mammary fat pad of 8-week-old BALB/C nude mice (Charles River Laboratories
  • the NP solution was concentrated to 15 mg/mL with the total encapsulated siRNA concentration being approximately 150 ⁇ g/mL.
  • siRNA-containing NPs were directly injected into the tumor at an equivalent dose of approximately 0.4 mg/kg entrapped siRNA. Right before dosing, the mice were monitored using an IVIS Spectrum bioluminescent and fluorescent imaging system (Caliper Life Sciences).
  • bioluminescence images were then taken daily for 3 days post-injection.
  • the bioluminescence intensity was analyzed using Livinglmage acquisition and analysis software. All of the in vivo imaging experiments were performed in quintuplicate.
  • Figure 5A is a graph showing qRT-PCR confirmation oiREVl and REV3L gene suppression using NY(si ' REVl, s ⁇ REV3L) in LNCaP human prostate adenocarcinoma cells at 24, 48 and 72 hr.
  • Figure 5B is a graph showing platinum dose-response curves (relative survival) in cells expressing normal [compound 1 in solution form, NP(compound 1)] or impaired
  • the experiment was conducted in quadruplicate (n 4). *Prior to treatment with the two siRNA - containing NP formulations, the cells were transfected with NP(siREVl, s REV3L) for 48 hours. The transfected cells were then treated with the two different formulations with escalating-dose of compound 1.
  • Figure 6 is a graph showing qRT-PCR confirmation ofREVl and REV3L gene suppression using NP(SLR£ 7, in MDA-MB-231 human breast carcinoma cells at 24, 48 and 72 hr.
  • Figure 8A is a graph showing qRT-PCR confirmation oiREVl and REV3 gene suppression in LNCaP cells that were harvested from xenograft tumor and isolated by GFP sorting 2 or 3 days post injection of NP(siR£Ti,
  • Figure 8B is a graph showing the inhibition of LNCaP xenograft tumor growth by (v) NP(siR£yi, s ⁇ REV3L, compound 1) in comparison with the following formulations: (i) saline, (ii) compound 1 in solution form, (iii) compound 1 encapsulated NP [NP(compound 1)], and (iv) NP(sL/?is F/, s ⁇ REV3L) with compound 1 in solution.
  • the dose of compound 1 and siRNA per injection was 4 mg kg and 0.4 mg/kg, respectively.
  • Figure 8C is a graph showing the survival curves of tumor-bearing mice treated with the aforementioned 5 formulations.
  • Day 0 represents the first day of W(&i ' REVl, siREV3L) administration.
  • PO.0136 Compound 1 vs NP(si&EF7, + Compound 1; P ⁇ 0.008 NP(Compound 1) vs NP(sLR£W, siREV3L, compound 1).
  • P values for all survival studies were determined using log-rank curve comparison tests.
  • *Prior to treatment with formulations (iv) and (v) the tumor-bearing mice were injected on day 0 and day 2 with NP(sLR£yi, Starting from the 4th day, the mice received intratumoral injections of the aforementioned 5 formulations twice weekly.
  • Day 0 represents the first day of NV(siREVl, s ⁇ REV3L) administration.
  • Figure 9A is a graph showing inhibition of LNCaP xenograft tumor growth by (iii) W(s REVl, s ⁇ REV3L, compound 1) in comparison with (i) saline and (ii) NP(scrambled siRNA, compound 1). The dose of compound 1 and siRNA per injection was 4 mg/kg and 0.4 mg kg, respectively.
  • Figure 9B is a graph showing survival curves of tumor-bearing mice treated with the aforementioned 3 formulations. Day 0 represents the first day of NP(sii?£ ' i, or NP(scrambled siRNA) administration.
  • Figure 10 is a graph shows the AAS analysis of platinum content within LNCaP cells treated with an equivalent dose of compound 1 either in solution or NP form.
  • Hydrophobic cationic material refers to a molecule containing a hydrophobic moiety covalently bound to a cationic moiety.
  • the cationic moiety can contain a single cationic site (e.g., small molecule cationic moiety) or a plurality of cationic sites (e.g., small molecule, oligomer, polymer, lipids, or dendrimer).
  • Amphiphilic material refers to a material containing a hydrophobic or more hydrophobic oligomer or polymer (e.g.,
  • biodegradable oligomer or polymer and a hydrophilic or more hydrophilic oligomer or polymer.
  • Olemer generally refers to molecules having up to 10 repeat units.
  • Polymer as used herein, generally refers to molecules having more than 10 repeat units.
  • subject refers to any organism to which the particles may be administered, e.g. for experimental, therapeutic, diagnostic, and/or prophylactic purposes.
  • Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.
  • treating can include preventing or alleviating one or more symptoms of a disease, disorder or condition from occurring in an animal which may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it; inhibiting one or more symptoms of the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition.
  • Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.
  • bioactive agent and “active agent”, as used
  • a bioactive agent is a substance used for the treatment (e.g., therapeutic agent), prevention (e.g., prophylactic agent), diagnosis (e.g., diagnostic agent), cure or mitigation of disease or illness, a substance which affects the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a predetermined physiological environment.
  • sufficient and “effective”, as used interchangeably herein, refer to an amount (e.g. mass, volume, dosage, concentration, and/or time period) needed to achieve one or more desired result(s).
  • therapeutically effective amount refers to that amount of the compound being administered which will relieve to some extent one or more of the symptoms of the disorder being treated.
  • biocompatible refers to a material that along with any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause any significant adverse effects to the recipient.
  • biocompatible materials are materials which do not elicit a significant inflammatory or immune response when administered to a patient.
  • pharmaceutically acceptable refers to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio, in accordance with the guidelines of agencies such as the Food and Drug Administration.
  • pharmaceutically acceptable carrier refers to all components of a pharmaceutical formulation which facilitate the delivery of the composition in vivo.
  • a “pharmaceutical composition” refers to a mixture of one or more of the compounds, or pharmaceutically acceptable salts thereof, with other chemical components, such as physiologically acceptable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • small molecule generally refers to an organic molecule that is less than about 2000 g/mol in molecular weight, less than about 1500 g/mol, less than about 1000 g/mol, less than about 800 g/mol, or less than about 500 g/mol. Small molecules are non-polymeric and/or non-oligomeric.
  • prodrug refers to an agent, including nucleic acids and proteins, which is converted into a biologically active form in vitro and/or in vivo.
  • Prodrugs are often useful because, in some situations, they may be easier to administer than the parent compound. They may, for instance, be bioavailable by oral administration whereas the parent compound is not.
  • the prodrug may also have improved solubility in pharmaceutical compositions over the parent drug.
  • a prodrug may be converted into the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis. Harper, N.J. (1962). Drug Latentiation in Jucker, ed. Progress in Drug Research, 4:221-294; Morozowich et al. (1977). Application of Physical Organic Principles to Prodrug Design in E. B.
  • molecular weight generally refers to the mass or average mass of a material. If a polymer or oligomer, the molecular weight can refer to the relative average chain length or relative chain mass of the bulk polymer. In practice, the molecular weight of polymers and oligomers can be estimated or characterized in various ways including gel permeation chromatography (GPC) or capillary viscometry. GPC molecular weights are reported as the weight-average molecular weight (M w ) as opposed to the number-average molecular weight (M n ). Capillary viscometry provides estimates of molecular weight as the inherent viscosity determined from a dilute polymer solution using a particular set of concentration, temperature, and solvent conditions.
  • copolymer generally refers to a single polymeric material that is comprised of two or more different monomers.
  • the copolymer can be of any form, such as random, block, graft, etc.
  • the copolymers can have any end-group, including capped or acid end groups.
  • biodegradable generally refers to a material that will degrade or erode under physiologic conditions to smaller units or chemical species that are capable of being metabolized, eliminated, or excreted by the subject.
  • the degradation time is a function of composition and morphology. Degradation times can be from hours to weeks.
  • hydrophilic refers to substances that have strongly polar groups that readily interact with water.
  • hydrophobic refers to substances that lack an affinity for water; tending to repel and not absorb water as well as not dissolve in or mix with water.
  • lipophilic refers to compounds having an affinity for lipids.
  • mean particle size generally refers to the statistical mean particle size (diameter) of the particles in the composition.
  • the diameter of an essentially spherical particle may be referred to as the physical or hydrodynamic diameter.
  • the diameter of a non-spherical particle may refer preferentially to the hydrodynamic diameter.
  • the diameter of a non-spherical particle may refer to the largest linear distance between two points on the surface of the particle.
  • Mean particle size can be measured using methods known in the art, such as dynamic light scattering.
  • Two populations can be said to have a "substantially equivalent mean particle size" when the statistical mean particle size of the first population of nanoparticles is within 20% of the statistical mean particle size of the second population of nanoparticles; more preferably within 15%, most preferably within 10%.
  • a monodisperse distribution refers to particle distributions in which 90% of the distribution lies within 5% of the mean particle size.
  • nucleic acid is a term of art that refers to a string of at least two base-sugar-phosphate combinations.
  • a polynucleotide contains more than 120 monomeric units since it must be distinguished from an oligonucleotide.
  • a polynucleotide contains 2 or more monomeric units. Nucleotides are the monomeric units of nucleic acid polymers.
  • nucleic acids refers to a string of at least two base-sugar- phosphate combinations. Natural nucleic acids have a phosphate backbone, artificial nucleic acids may contain other types of backbones, but contain the same bases. Nucleotides are the monomeric units of nucleic acid polymers.
  • RNA includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • RNA may be in the form of an tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti- sense RNA, RNAi, siRNA, and ribozymes.
  • PNAs peptide nucleic acids
  • phosphorothioates and other variants of the phosphate backbone of native nucleic acids.
  • siRNA means a small inhibitory ribonucleic acid.
  • the siRNA are typically less than 30 nucleotides in length and can be single or double stranded.
  • the ribonucleotides can be natural or artificial and can be chemically modified.
  • Longer siRNAs can comprise cleavage sites that can be enzymatically or chemically cleaved to produce siRNAs having lengths less than 30 nucleotides, typically 21 to 23 nucleotides.
  • siRNAs share sequence homology with corresponding target mRNAs. The sequence homology can be 100 percent or less but sufficient to result is sequence specific association between the siRNA and the targeted mRNA. Exemplary siRNAs do not activate the interferon signal transduction pathway.
  • inhibitory nucleic acid means an RNA, DNA, or combination thereof that interferes or interrupts the translation of mRNA. Inhibitory nucleic acids can be single or double stranded. The nucleotides of the inhibitory nucleic acid can be chemically modified, natural or artificial. II. Nanoparticles
  • Nanoparticles containing an aqueous core containing one or more nucleic acids, such as siRNA, and a shell containing one or more hydrophobic cationic materials, one or more amphiphilic materials, and one or more therapeutic, diagnostic, and/or prophylactic agents are.
  • the hydrophobic cationic material and the hydrophobic portion of the amphiphilic material provide a non-polar polymer matrix for loading non- polar drugs, protect and promoting siRNA molecule retention inside the NP core, and control drug release.
  • the hydrophilic portion of the amphiphilic material can form a corona around the particle which prolongs circulation of the particles in the blood stream and decreases uptake by the RES.
  • the shell of the particles contains one or more amphiphilic materials.
  • the amphiphilic material contains one or more biodegradable oligomeric or polymeric segments or blocks and one or more hydrophilic oligomeric or polymeric segments or blocks.
  • the biodegradable oligomeric or polymeric segment(s) or block(s) is hydrophobic.
  • the amphiphilic material is a hydrophobic, biodegradable polymer terminated with a hydrophilic block.
  • Biodegradable polymers can include polymers that are insoluble or sparingly soluble in water that are converted chemically or enzymatically in the body into water-soluble materials.
  • Biodegradable polymers can include soluble polymers crosslinked by hydrolyzable cross-linking groups to render the crosslinked polymer insoluble or sparingly soluble in water.
  • Biodegradable polymers in the shell include, but are not limited to, polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polylactides, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose tri
  • biodegradable polymers include, but are not limited to, polyesters, poly(ortho esters), poly(ethylene imines), poly(caprolactones), poly(hydroxybutyrates), poly(hydroxyvalerates), polyanhydrides, poly(acrylic acids), polyglycolides, poly(urethanes), polycarbonates, polyphosphate esters, polyphosphazenes, derivatives thereof, linear and branched copolymers and block copolymers thereof, and blends thereof.
  • the polymeric core contains
  • biodegradable polyesters such as poly(lactic acid), poly(glycolic acid), and poly(lactic-co-glycolic acid).
  • the biodegradable polymer in the shell is a hydrophobic biodegradable polymer.
  • suitable hydrophobic polymers include, but are not limited to, polyhydroxyacids such as poly(lactic acid), poly(glycolic acid), and poly(lactic acid-co-glycolic acids); polyhydroxyalkanoates such as poly3-hydroxybutyrate or poly4- hydroxybutyrate; polycaprolactones; poly(orthoesters); polyanhydrides; poly(phosphazenes); poly(lactide-co-caprolactones); polycarbonates such as tyrosine polycarbonates; polyamides (including synthetic and natural polyamides), polypeptides, and poly(amino acids); polyesteramides;
  • polyesters poly(dioxanones); poly(alkylene alkylates); hydrophobic polyethers; polyurethanes; polyetheresters; polyacetals; polycyanoacrylates; polyacrylates; polymethylmethacrylates; polysiloxanes;
  • polyphosphates polyhydroxyvalerates; polyalkylene oxalates; polyalkylene succinates; poly(maleic acids), as well as copolymers thereof.
  • the polymeric core contains biodegradable polyesters or polyanhydrides such as poly(lactic acid), poly(glycolic acid), and poly(lactic-co-glycolic acid).
  • the molecular weight of the biodegradable oligomeric or polymeric segment can be varied to tailor the properties of polymeric particle shell.
  • the molecular weight of the biodegradable polymer can be varied to engineer nanoparticles possessing the required average particle size and degradation profile.
  • the hydrophobic polymer segment has a molecular weight of between about 150 Da and about lOOkDa, more preferably between about 1 kDa and about 75 kDa, most preferably between about 5 kDa and about 50 kDa.
  • the amphiphilic material also contains one or more hydrophilic segments or blocks.
  • the hydrophilic segment(s) or block(s) function as stealth polymers in order to prolong circulation of the particles and avoid uptakes by the RES.
  • Suitable stealth polymers include, but are not limited to, homo polymers or copolymers of polyalkene glycols, such as poly(ethylene glycol), poly(propylene glycol), poly(butylene glycol), and acrylates and acrylamides, such as hydroxyethyl methacrylate and hydroxypropyl-methacrylamide.
  • the hydrophilic polymer segment typically has a molecular weight of between about 150 Da and about 20kDa, more preferably between about 500 Da and about 10 kDa, most preferably between about 1 kDa and about 5 kDa.
  • the amphiphilic material contains one or more
  • PLA or PLGA covalently bound to one or more blocks of PEG.
  • the shell of the particles also contains a hydrophobic cationic
  • the cationic material is a material that is
  • the cationic under physiological conditions.
  • the cationic is cationic
  • amine containing moieties such as amine
  • amine-containing polymers such as PEI
  • the cationic moieties are functionalized with one or more
  • hydrophobic/lipid moieties such as lipophilic alkyl chains (e.g., C6-C30,
  • hydrophobic groups which can be coupled to obtain hydrophobic cationic
  • the cationic moiety promotes retention of the siRNA in the core through electrostatic interaction while the hydrophobic moiety provides controlled release of the siRNA as well as any active agents in the shell.
  • Therapeutic, Prophylactic, and Diagnostic Agents Any therapeutic, prophylactic, or diagnostic agent can be any therapeutic, prophylactic, or diagnostic agent.
  • the shell contains a therapeutic, prophylactic, or diagnostic agent.
  • the core contains a therapeutic, prophylactic, or diagnostic agent in addition to the siRNA.
  • the shell and core contain a therapeutic, prophylactic, or diagnostic agent.
  • the shell contains one or more non-polar therapeutic, prophylactic, and/or diagnostic agent
  • the loading range for the agent within the particles is from about 0.01 to about 80% (agent weight/particle weight), preferably from 0.01 % to about 50%) (wt/wt), more preferably from about 0.01% to about 25% (wt/wt), even more preferably from about 0.01% to about 10% (wt/wt), most preferably from about 0.1% to about 5% (wt/wt).
  • the percent loading is typically from about 0.01 ) to about 20%) (wt/wt), although higher loadings may be achieved for hydrophobic drugs and/or insoluble metals.
  • typical loadings are from about 0.01 % to about 10% (wt/wt), preferably from about 0.01%) to about 5.0%) (wt/wt), more preferably from about 0.01% to about 2.5% (wt/wt), most preferably from about 0.01% to about 1% (wt/wt).
  • the loading can be calculated relative to the mass of the polymer, lipid, or inorganic particles.
  • the particles can include one or more chemotherapeutic agents for the treatment of cancer and other diseases.
  • the chemotherapeutic agent is hydrophobic and therefore is dispersed within the more hydrophobic shell of the particles.
  • the biodegradable polymer and/or hydrophobic cationic material in the shell provide controlled release of the chemotherapeutic agent.
  • Chemotherapeutics includes, but not limited to, the following classes: alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, monoclonal antibodies, and other anti-tumor agents.
  • tyrosine kinase inhibitor imatinib mesylate (Gleevec® or Glivec®)
  • cisplatin carboplatin, oxaliplatin, mechloethamine, cyclophosphamide, chlorambucil, azathioprine, mercaptopurine, pyrimidine, vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin (L01CB), etoposide, docetaxel, topoisomerase inhibitors (L01CB and L01XX) irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, dactinomycin, lonidamine, and monoclonal antibodies, such as trastuzumab
  • Additional exemplary active agents include PARP inhibitors, survivin inhibitors, estradiol, and dichloroacetate.
  • active agents include, but are not limited to, antimicrobial agents, analgesics, anti-inflammatory agents, and other chemotherapeutic or anti-cancer agents.
  • Antibiotics can be incorporated into the particle, such as vancomycin, which is frequently used to treat infections, including those due to methicillin resistant staph aureus (MRSA).
  • MRSA methicillin resistant staph aureus
  • the particle optionally includes cyclosporin, a lipophilic drug that is an immunosuppressant agent, widely used post-allogeneic organ transplant to reduce the activity of the patient's immune system and the risk of organ rejection (marketed by Novartis under the brand names Sandimmune®, the original formulation, and Neoral® for the newer microemulsion
  • Particles comprising cyclosporine can be used in topical emulsions for treating keratoconjunctivitis sicca, as well.
  • particles with multifunctional surface domains incorporating such drugs can be designed to deliver equivalent dosages of the various drugs directly to the cancer cells, thus potentially minimizing the amount delivered generally to the patient and minimizing collateral damage to other tissues.
  • the chemotherapeutic is a platinum-based chemotherapeutic or prodrug thereof.
  • the chemotherapeutic is cisplatin or a prodrug thereof.
  • the drug can be dispersed (e.g., non-covalently associated) in the polymeric shell or can be covalently bound to the polymeric shell.
  • chemotherapeutic agent can be conjugated to a biodegradable polymer.
  • a cisplatin prodrug platinum monosuccinate
  • the oxaliplatin prodrug can be synthesized with carboxyl groups at the axial position, which will be coupled to the PLA-OH.
  • Polylactide with paclitaxel pendant groups was prepared by generating carboxyl group containing polylactide by treating PLA-OH with succinic anhydride, and this compound was coupled directly with hydroxyl groups of paclitaxel.
  • tubacin-functionalized polymers the same carboxyl group containing polylactides can be conjugated to the hydroxyl groups of tubacin.
  • the particles contain an aqueous core surrounded by the more hydrophobic shell.
  • the aqueous core contains one or more nucleic acids, such as siRNAs, shRNA, DNA antisense, DNA plasmid, peptides, therapeutic proteins, water soluble small drugs, or the mixture of those agents.
  • the inhibitory nucleic acids of certain embodiments are directed to target genes encoding proteins involved in DNA repair and the acquisition of multidrug resistance (MDR), for example in the treatment of cancer.
  • the inhibitory nucleic acids disclosed herein include small inhibitory ribonucleic acids (siRNAs) that are typically less than 30 nucleotides in length, more typically 21 to 23 nucleotides in length, and can be single or double stranded.
  • One strand of a double-stranded siRNA comprises at least a partial sequence complementary to a target mRNA.
  • the ribonucleotides of the siRNA can be natural or artificial and can be chemically modified.
  • siRNAs can comprise cleavage sites that can be enzymatically or chemically cleaved to produce siRNAs having lengths less than 30 nucleotides.
  • siRNAs share sequence homology with corresponding target mRNAs.
  • the phosphate backbones of the siRNAs can be chemically modified to resist enzymatic degradation.
  • the sequence homology can be about 100 percent or less, but sufficient to result is sequence specific association between the siRNA and the targeted mRNA.
  • RNA interference RNA interference
  • dsRNA double-stranded RNA
  • RNAi small interfering RNAs
  • siRNA duplexes bind to a nuclease complex to form what is known as the RNA-induced silencing complex, or RISC.
  • RISC RNA-induced silencing complex
  • An ATP- depending unwinding of the siRNA duplex is required for activation of the RISC.
  • the active RISC targets the homologous transcript by base pairing interactions and cleaves the mRNA -12 nucleotides from the 3' terminus of the siRNA.
  • the siRNAs can form a complex with additional proteins and/or cofactors to enzymatically cleave a target mRNA.
  • the particles such as the surface of the particles, can be modified to facilitate targeting through the attachment of targeting molecules.
  • Exemplary target molecules include proteins, peptides, nucleic acids, lipids, saccharides, or polysaccharides that bind to one or more targets associated with an organ, tissue, cell, or extracellular matrix, or specific type of tumor or infected cell.
  • the degree of specificity with which the particles are targeted can be modulated through the selection of a targeting molecule with the appropriate affinity and specificity.
  • a targeting moiety can be a polypeptide, such as an antibody that specifically recognizes a tumor marker that is present exclusively or in higher amounts on a malignant cell (e.g., a tumor antigen).
  • Suitable targeting molecules that can be used to direct nanoparticles to cells and tissues of interest, as well as methods of conjugating target molecules to nanoparticles, are known in the art.
  • Targeting molecules can also include neuropilins and endothelial targeting molecules, integrins, selectins, and adhesion molecules. Targeting molecules can be covalently bound to particles using a variety of methods known in the art. In some embodiments, the targeting moieties are covalently associated with the polymer, preferably via a linker cleaved at the site of delivery.
  • the nanoparticles can contain one or more polymer conjugates containing end-to-end linkages between the polymer and a targeting element or a detectable label.
  • a modified polymer can be a PLGA- PEG-peptide block polymer.
  • targeting moieties include peptides such as iRGD, LyPl ; small molecule such as folate, aptamers and antibodies or their combinations at various molar ratios.
  • the targeting element of the nanoparticle can be an antibody or antigen binding fragment thereof.
  • the targeting elements should have an affinity for a cell-surface receptor or cell-surface antigen on the target cells and result in internalization of the particle within the target cell.
  • the targeting element can specifically recognize and bind to a target molecule specific for a cell type, a tissue type, or an organ.
  • the target molecule can be a cell surface polypeptide, lipid, or glycolipid.
  • the target molecule can be a receptor that is selectively expressed on a specific cell surface, a tissue or an organ.
  • Cell specific markers can be for specific types of cells including, but not limited to stem cells, skin cells, blood cells, immune cells, muscle cells, nerve cells, cancer cells, virally infected cells, and organ specific cells.
  • the cell markers can be specific for endothelial, ectodermal, or mesenchymal cells. Representative cell specific markers include, but are not limited to cancer specific markers.
  • Additional targets that can be recognized by the targeting element include VEGF/KDR, Tie2, vascular cell adhesion molecule (VCAM), endoglin and ⁇ 5 ⁇ 3 integrin vitronectin.
  • the targeting peptides can be covalently associated with the polymer of the outer shell and the covalent association can be mediated by a linker.
  • the targeting element specifically binds to an antigen that is expressed by tumor cells.
  • the antigen expressed by the tumor may be specific to the tumor, or may be expressed at a higher level on the tumor cells as compared to non-tumor cells.
  • Antigenic markers such as serologically defined markers known as tumor associated antigens, which are either uniquely expressed by cancer cells or are present at markedly higher levels (e.g., elevated in a statistically significant manner) in subjects having a malignant condition relative to appropriate controls, are contemplated for use in certain embodiments.
  • Tumor-associated antigens may include, for example, cellular oncogene-encoded products or aberrantly expressed proto-oncogene-encoded products (e.g., products encoded by the neu, ras, trk, and kit genes), or mutated forms of growth factor receptor or receptor-like cell surface molecules (e.g., surface receptor encoded by the c-erb B gene).
  • Other tumor- associated antigens include molecules that may be directly involved in transformation events, or molecules that may not be directly involved in oncogenic transformation events but are expressed by tumor cells (e.g., carcinoembryonic antigen, CA-125, melonoma associated antigens, etc.) (see, e.g., U.S. Pat. No.
  • Genes that encode cellular tumor associated antigens include cellular oncogenes and proto-oncogenes that are aberrantly expressed.
  • cellular oncogenes encode products that are directly relevant to the transformation of the cell, and because of this, these antigens are particularly preferred targets for immunotherapy.
  • An example is the tumorigenic neu gene that encodes a cell surface molecule involved in oncogenic
  • proto-oncogenes the normal genes which are mutated to form oncogenes
  • proto-oncogenes may be aberrantly expressed (e.g., overexpressed), and this aberrant expression can be related to cellular transformation.
  • proto-oncogenes can be targeted.
  • Some oncogenes encode growth factor receptor molecules or growth factor receptor-like molecules that are expressed on the tumor cell surface.
  • An example is the cell surface receptor encoded by the c-erbB gene.
  • Other tumor-associated antigens may or may not be directly involved in malignant transformation. These antigens, however, are expressed by certain tumor cells and may therefore provide effective targets.
  • Some examples are carcinoembryonic antigen (CEA), CA 125 (associated with ovarian carcinoma), and melanoma specific antigens.
  • tumor associated antigens are detectable in samples of readily obtained biological fluids such as serum or mucosal secretions.
  • One such marker is CA125, a carcinoma associated antigen that is also shed into the bloodstream, where it is detectable in serum (e.g., Bast, et al., N. Eng. J. Med., 309:883 (1983); Lloyd, et al., Int. J. Cane, 71 :842 (1997).
  • CA125 levels in serum and other biological fluids have been measured along with levels of other markers, for example, carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC), tissue polypeptide specific antigen (TPS), sialyl TN mucin (STN), and placental alkaline phosphatase (PLAP), in efforts to provide diagnostic and/or prognostic profiles of ovarian and other carcinomas (e.g., Sarandakou, et al., Acta Oncol, 36:755 (1997); Sarandakou, et al., Eur. J. Gynaecol. Oncol, 19:73 (1998); Meier, et al., Anticancer Res.
  • CEA carcinoembryonic antigen
  • SCC squamous cell carcinoma antigen
  • TPS tissue polypeptide specific antigen
  • STN sialyl TN mucin
  • PLAP placental alkaline phosphatase
  • Elevated serum CA125 may also accompany neuroblastoma (e.g., Hirokawa, et al, Surg. Today, 28:349 (1998), while elevated CEA and SCC, among others, may accompany colorectal cancer (Gebauer, et al., Anticancer Res., 17(4B):2939 (1997)).
  • mesothelin is detectable only as a cell-associated tumor marker and has not been found in soluble form in serum from ovarian cancer patients, or in medium conditioned by OVCAR-3 cells (Chang, et al., Int. J. Cancer, 50:373 (1992)).
  • Structurally related human mesothelin polypeptides also include tumor-associated antigen polypeptides such as the distinct mesothelin related antigen (MRA) polypeptide, which is detectable as a naturally occurring soluble antigen in biological fluids from patients having malignancies (see WO 00/50900).
  • MRA mesothelin related antigen
  • a tumor antigen may include a cell surface molecule.
  • Tumor antigens of known structure and having a known or described function include the following cell surface receptors: HER1 (GenBank Accession No. U48722), HER2 (Yoshino, et al., J. Immunol, 152:2393 (1994); Disis, et al., Cane. Res., 54: 16 (1994); GenBank Acc. Nos. X03363 and M17730), HER3 (GenBank Acc. Nos. U29339 and M34309), HER4 (Plowman, et al., Nature, 366:473 (1993); GenBank Acc. Nos.
  • EGFR epidermal growth factor receptor
  • vascular endothelial cell growth factor GenBank No. M32977
  • vascular endothelial cell growth factor receptor GenBank Acc. Nos. AF022375, 1680143, U48801 and X62568
  • insulin-like growth factor-I GenBank Acc. Nos. X00173, X56774, X56773, X06043, European Patent No. GB
  • insulin-like growth factor-II GenBank Acc. Nos. X03562, X00910, M17863 and M17862
  • transferrin receptor Trowbridge and Omary, Proc. Nat. Acad. USA, 78:3039 (1981); GenBank Acc. Nos. X01060 and Ml 1507), estrogen receptor (GenBank Acc. Nos. M38651, X03635, X99101, U47678 and M12674), progesterone receptor (GenBank Acc. Nos. X51730, X69068 and M15716), follicle stimulating hormone receptor (FSH- R) (GenBank Acc. Nos.
  • retinoic acid receptor GenBank Acc. Nos. L12060, M60909, X77664, X57280, X07282 and X06538), MUC-1 (Barnes, et al, Proc. Nat. Acad. Sci. USA, 86:7159 (1989); GenBank Acc. Nos. M65132 and M64928) NY-ESO-1 (GenBank Acc. Nos. AJ003149 and U87459), NA 17-A (PCT Publication No. WO 96/40039), Melan- A/MART- 1 (Kawakami, et al., Proc. Nat. Acad. Sci.
  • GenBank Acc. Nos. U06654 and U06452 GenBank Acc. Nos. U06654 and U06452
  • tyrosinase Topalian, et al., Proc. Nat. Acad. Sci. USA, 91 :9461 (1994); GenBank Acc. No. M26729; Weber, et al., J. Clin. Invest, 102: 1258 (1998)), Gp-100 (Kawakami, et al., Proc. Nat. Acad. Sci. USA, 91 :3515 (1994); GenBank Acc. No. S73003, Adema, et al, /. Biol.
  • any of the CTA class of receptors including in particular HOM- MEL-40 antigen encoded by the SSX2 gene (GenBank Acc. Nos. X86175, U90842, U90841 and X86174), carcinoembryonic antigen (CEA, Gold and Freedman, J. Exp. Med., 121:439 (1985); GenBank Acc. Nos. M59710, M59255 and M29540), and PyLT (GenBank Acc. Nos. J02289 and J02038); p97 (melanotransferrin) (Brown, et al, J. Immunol, 127:539-46 (1981);
  • PSA prostate surface antigen
  • ⁇ -human chorionic gonadotropin ⁇ -HCG ⁇ -human chorionic gonadotropin ⁇ -HCG
  • melanoma antigen gp75 (Vijayasardahi, et al, J. Exp. Med., 171: 1375-80 (1990); GenBank Accession No. X51455); human cytokeratin 8; high molecular weight melanoma antigen (Natali, et al,
  • CT antigens of interest include antigens regarded in the art as "cancer/testis” (CT) antigens that are immunogenic in subjects having a malignant condition (Scanlan, et al., Cancer Immun., 4: 1 (2004)).
  • CT antigens include at least 19 different families of antigens that contain one or more members and that are capable of inducing an immune response, including but not limited to MAGEA (CT1); BAGE (CT2); MAGEB (CT3); GAGE (CT4); SSX (CT5); NY-ESO- 1 (CT6); MAGEC (CT7); SYCP 1 (C8); SPANXB 1 (CT11.2); NA88 (CT18); CTAGE (CT21); SPA 17 (CT22); OY- TES-1 (CT23); CAGE (CT26); HOM-TES-85 (CT28); HCA661 (CT30); NY-SAR-35 (CT38); FATE (CT43); and TPTE (CT44).
  • CT1 MAGEA
  • Additional tumor antigens that can be targeted include, but not limited to, alpha- actinin-4, Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR- fucosyltransferaseAS fusion protein, HLA-A2, HLA-A1 1, hsp70-2, KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP, myosin class I, OS-9, pml- RARa fusion protein, PTPRK, K-ras, N-ras, Trios ephosphate isomeras, Bage-1, Gage 3,4,5,6,7, GnTV, Herv-K-mel, Lü-1, Mage- Al,2,3,4,6,10, 12, Mage-C2, NA-
  • the targeting element is a peptide.
  • the plaque targeted peptide can be, but is not limited to, one or more of the following: RGD, iRGD(CRGDK/RGPD/EC) (SEQ ID NO: l), LyP-1, P3 (CKGGRAKDC) (SEQ ID NO:2), or their combinations at various molar ratios.
  • RGD iRGD(CRGDK/RGPD/EC)
  • LyP-1 LyP-1
  • P3 CKGGRAKDC
  • the targeting peptides can be covalently associated with the polymer and the covalent association can be mediated by a linker.
  • the targeting element can be an antibody or an antigen-binding fragment thereof.
  • the antibody can be any type of immunoglobulin that is known in the art.
  • the antibody can be of any isotype, e.g., IgA, IgD, IgE, IgG, IgM, etc.
  • the antibody can be monoclonal or polyclonal.
  • the antibody can be a naturally-occurring antibody, e.g., an antibody isolated and/or purified from a mammal, e.g., mouse, rabbit, goat, horse, chicken, hamster, human, etc.
  • the antibody can be a genetically- engineered antibody, e.g., a humanized antibody or a chimeric antibody.
  • the antibody can be in monomeric or polymeric form.
  • the antigen binding portion of the antibody can be any portion that has at least one antigen binding site, such as Fab, F(ab') 2 , dsFv, sFv, diabodies, and triabodies.
  • the antibody is a single chain antibody.
  • Aptamers are oligonucleotide or peptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Aptamers bind to targets such as small organics, peptides, proteins, cells, and tissues. Unlike antibodies, some aptamers exhibit stereoselectivity. The aptamers can be designed to bind to specific targets expressed on cells, tissues or organs.
  • the particles may have any diameter.
  • the particles can have a diameter of about 10 nm to about 10 microns, about 10 nm to about 1 micron, about 10 nm to about 500 nm, about 20 nm to about 500 nm, or about 25 nm to about 250 nm.
  • the particle is a nanoparticle having a diameter from about 25 nm to about 250 nm.
  • the particles are nanoparticles having a diameter from about 180 nm to about 250 nm, preferably from about 180 nm to about 230 nm.
  • the polydispersity is from about 0.05 to 0.30, preferably from about 0.05 to about 0.25, more preferably from about 0.05 to about 0.20, more preferably from about 0.05 to about 0.15, most preferably from about 0.05 to about 0.10.
  • the particles may have any zeta potential.
  • the particles can have a zeta potential from -300 mV to + 300 mV, -100 mV to +100 mV, from -50 mV to +50 mV, from -40 mV to +40 mV, from -30 mV to +30 mV, from -20 mV to +20 mV, from -10 mV to +10mV, or from -5mV to +5 mV.
  • the particles can have a negative or positive zeta potential.
  • the particles have a substantially neutral zeta potential, i.e. the zeta potential is approximately 0 mV.
  • the particles have a zeta potential of approximately -30 to about 30 mV, preferably from about -20 to about 20 mV, more preferably from about -10 to about 10 mV.
  • the particles can be formulated with appropriate pharmaceutically acceptable carries to into pharmaceutical compositions for administration to an individual in need thereof.
  • the formulations can be administered enterally (e.g., oral) or parenterally (e.g., by injection or infusion). Other routes of administration include, but are not limited to, transdermal.
  • parenteral administration means administration by any method other than through the digestive tract or non-invasive topical or regional routes.
  • parenteral administration may include administration to a patient intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly,
  • intratumorally intramuscularly, subcutaneous ly, subconjunctivally, intravesicularly, intrapericardially, intraumbilically, by injection, and by infusion.
  • Parenteral formulations can be prepared as aqueous compositions using techniques is known in the art.
  • such compositions can be prepared as injectable formulations, for example, solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w/o) emulsions, oil-in-water (o/w) emulsions, and
  • microemulsions thereof, liposomes, or emulsomes are examples of microemulsions thereof, liposomes, or emulsomes.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and combinations thereof.
  • polyols e.g., glycerol, propylene glycol, and liquid polyethylene glycol
  • oils such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.)
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants.
  • isotonic agents for example, sugars or sodium chloride.
  • Solutions and dispersions of the active compounds as the free acid or base or pharmacologically acceptable salts thereof can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, viscosity modifying agents, and combination thereof.
  • Suitable surfactants may be anionic, cationic, amphoteric or nonionic surface active agents.
  • Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions.
  • anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2- ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate.
  • Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine.
  • nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG- 1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether,
  • Poloxamer® 401 stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide.
  • amphoteric surfactants include sodium N-dodecyl-.beta.-alanine, sodium N-lauryl-.beta.-iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.
  • the formulation can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal.
  • the formulation may also contain an antioxidant to prevent degradation of the active agent(s).
  • the formulation is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution.
  • Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.
  • Water soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.
  • Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the powders can be prepared in such a manner that the particles are porous in nature, which can increase dissolution of the particles. Methods for making porous particles are well known in the art.
  • Enteral formulations are prepared using pharmaceutically acceptable carriers.
  • carrier includes, but is not limited to, diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof.
  • Polymers used in the dosage form include hydrophobic or hydrophilic polymers and pH dependent or independent polymers. Preferred hydrophobic and hydrophilic polymers include, but are not limited to, hydroxypropyl methylcellulose,
  • hydroxypropyl cellulose hydroxyethyl cellulose, carboxy methylcellulose, polyethylene glycol, ethylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl acetate, and ion exchange resins.
  • Carrier also includes all components of the coating composition which may include plasticizers, pigments, colorants, stabilizing agents, and glidants.
  • Formulations can be prepared using one or more pharmaceutically acceptable excipients, including diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof.
  • Delayed release dosage formulations can be prepared as described in standard references such as "Pharmaceutical dosage form tablets”, eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989), "Remington - The science and practice of pharmacy", 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and "Pharmaceutical dosage forms and drug delivery systems", 6th Edition, Ansel et al., (Media, PA: Williams and Wilkins, 1995). These references provide information on excipients, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules. These references provide information on carriers, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules.
  • Stabilizers are used to inhibit or retard drug decomposition reactions which include, by way of example, oxidative reactions.
  • Suitable stabilizers include, but are not limited to, antioxidants, butylated hydroxytoluene (BHT); ascorbic acid, its salts and esters; Vitamin E, tocopherol and its salts; sulfites such as sodium metabisulphite; cysteine and its derivatives; citric acid; propyl gallate, and butylated hydroxyanisole (BHA).
  • hydrophobic cationic moieties can be prepared using techniques known in the art.
  • amine containing small molecules, polymers, (e.g., PEI), and/or dendrimers can be functionalized with one or more hydrophobic groups, such as lipophilic alkyl groups, cholesterol, or combinations thereof. These groups can be introduced by reaction the amine with the reactive derivative of the hydrophobic moiety, such as
  • chloroformate acid chloride
  • acrylates acrylamides
  • epoxide-terminated compounds epoxide-terminated compounds
  • the chemotherapeutic agent is covalently bound to the amphiphilic material used to form the particle shell.
  • Polymer- drug conjugates can be prepared using synthetic methods known in the art. The appropriate route for synthesis of a given polymer-drug conjugate can be determined in view of a number of factors, such as the structure of the polymer-drug conjugate, the composition of the polymer segments which make up the polymer-drug conjugate, the identity of the one or more drugs attached to the polymer-drug conjugate, as well as the structure of the conjugate and its components as it relates to compatibility of functional groups, protecting group strategies, and the presence of labile bonds.
  • microencapsulation techniques include, but are not limited to, spray drying, interfacial polymerization, hot melt encapsulation, phase separation encapsulation (spontaneous emulsion microencapsulation, solvent evaporation microencapsulation, and solvent removal microencapsulation), coacervation, low temperature microsphere formation, and phase inversion nanoencapsulation (PIN).
  • the particles are prepared using an emulsion- based technique.
  • the particles are prepared using a double emulsion solvent evaporation technique.
  • the amphiphilic material and the hydrophobic cationic material are dissolved in a suitable organic solvent, such as methylene chloride or dichloromethane (DCM), with or without a therapeutic agent.
  • DCM dichloromethane
  • the siRNA is reconstituted in purified water, such as HyPureTM molecular biology grade water (Hyclone Laboratories, Inc., Logan, Utah). The siRNA solution is added dropwise to the solution of the amphiphilic material and the hydrophobic cationic material and emulsified to form a first emulsion.
  • the emulsion is added to an aqueous solution of surfactant, such as PVA, to form a double emulsion.
  • the final emulsion is added to water and stirred for an extended period of time (e.g., 3 hours) to allow the organic solvent to evaporate and the particles to harden. Residual organic solvent and/or unencapsulated molecules are removed by washing.
  • an extended period of time e.g. 3 hours
  • phase separation microencapsulation techniques a polymer solution is stirred, optionally in the presence of one or more active agents to be encapsulated. While continuing to uniformly suspend the material through stirring, a nonsolvent for the polymer is slowly added to the solution to decrease the polymer's solubility. Depending on the solubility of the polymer in the solvent and nonsolvent, the polymer either precipitates or phase separates into a polymer rich and a polymer poor phase. Under proper conditions, the polymer in the polymer rich phase will migrate to the interface with the continuous phase, encapsulating the active agent(s) in a droplet with an outer polymer shell.
  • Spontaneous emulsification involves solidifying emulsified liquid polymer droplets formed above by changing temperature, evaporating solvent, or adding chemical cross-linking agents.
  • One or more active agents to be incorporated are optionally added to the solution, and the mixture is suspended in an aqueous solution that contains a surface active agent such as polyvinyl alcohol). The resulting emulsion is stirred until most of the organic solvent evaporated, leaving solid microparticles/nanoparticles. This method is useful for relatively stable polymers like polyesters and polystyrene.
  • Nanoparticles can also be formed using the phase inversion nanoencapsulation (PIN) method, wherein a polymer is dissolved in a "good” solvent, fine particles of a substance to be incorporated, such as a drug, are mixed or dissolved in the polymer solution, and the mixture is poured into a strong non solvent for the polymer, to spontaneously produce, under favorable conditions, polymeric microspheres, wherein the polymer is either coated with the particles or the particles are dispersed in the polymer.
  • PIN phase inversion nanoencapsulation
  • Nanoparticles can be prepared using microfluidic devices.
  • a polymeric material is mixed with a drug or drug combinations in a water miscible organic solvent.
  • the water miscible organic solvent can be one or more of the following: acetone, ethanol, methanol, isopropyl alcohol, acetonitrile and Dimethyl sulfoxide (DMSO).
  • DMSO Dimethyl sulfoxide
  • the resulting mixture solution is then added to an aqueous solution to yield nanoparticle solution.
  • the targeted peptides or fluorophores or drugs may be associated with the surface of, encapsulated within, surrounded by, and/or distributed throughout the polymeric matrix of the particles.
  • the particles can be used for the co-delivery of an effective amount of a nucleic acid, such as siRNA, and a therapeutic, diagnostic, and/or prophylactic agent.
  • a nucleic acid such as siRNA
  • the therapeutic, diagnostic, and/or prophylactic agent can be a small molecule, a biomolecule or macromolecules, or combinations thereof.
  • the particles contain one or more siRNA that target genes encoding proteins involved in DNA repair and/or the acquisition of multidrug resistance (MDR).
  • MDR multidrug resistance
  • the particles described here exhibit excellent encapsulation efficiency (EE%) of siRNA compared to the same nanoparticles without the hydrophobic cationic material.
  • the EE is at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 96, 98, or 99%.
  • particles lacking the hydrophobic cationic moiety had EE of about 6-10%.
  • the particles are able to release siRNA and a small molecule therapeutic in a controlled manner.
  • the particles release about 5, 8, 10, 12, 15, or 20% of the small molecule therapeutic over about 2, 3, 4, 5, or 6 hours, followed by sustained release over about 6, 7, 8, 9, 10, 11, or 12 hours.
  • the particles release about 75, 80, 85, 90, or 95% of the encapsulated small molecule therapeutic over about one week.
  • the particles can release about 20, 25, 30, 35, 40, 45, 50, 55, or 60% of the encapsulated siRNA over about 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours and about 75, 80, 85, 90, or 95% of the encapsulated siRNA over about 5, 6, 7, 8, 9, or 10 days.
  • Particles containing the siRNA siLuc showed increased silencing of luciferase expression as a function of concentration.
  • NP(siLuc) achieved greater than 95% luciferase knockdown, a more efficient silencing efficacy than the commercially available liposome-based lipoplex (Lipo2000-siRNA complex).
  • XTT 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H- Tetrazolium-5-Carboxanilide
  • the particles exhibited significant activity in inhibiting tumor growth.
  • the bioluminescence intensity from the control group mice increased rapidly from day 0 to day 3 with day 3 being a near 64% increase in intensity compared to day 0.
  • NP(siLuc) showed a drastic decrease in bioluminescence intensity one-day post injection and increased in the following days. An 80% decrease in luciferase expression in the treated group one day post injection.
  • the particles also exhibited sustained knockdown efficiency in a LNCaP tumor model.
  • a qRT-PCR assay revealed a sustained knockdown efficiency of up to 87% for both genes REV1 and REV3L in LNCaP cells over the course of three days.
  • a similar gene silencing efficacy was observed in MDA-MB-231 cells.
  • these developed NPs are capable of simultaneously targeting multiple genes.
  • NP(compound 1) [NP(compound 1)], (iii) NP(siREVl, siREV3L) with compound 1 in solution, and (iv) NP(siREVl, siREV3L, compound 1).
  • the cells Prior to treatment with the two siRNA-containing NP formulations, the cells were transfected with NP(siREVl, siREV3L) for 24 hours in order to achieve substantial levels of Rev 1 and Rev3L suppression. The transfected cells were then treated with the two different siRNA-containing formulations with escalating-dose of compound 1.
  • the first formulation consists of
  • NP(siREVl, siREV3L) with compound 1 in solution form, whereas the second contains compound 1 within the NP [NP(siREVl, siREV3L, compound 1)].
  • N-hydroxysuccinimide (NHS), l-ethyl-3-[3- dimethylaminopropyljcarbodiimide hydrochloride (EDC), 1,2- epoxytetradecane, ethylenediamine core-PAMAM generation 0 dendrimer, decanoic anhydride and cis,cis,trans-[Pt(NH 3 ) 2 Cl2(OH)2] were purchased from Aldrich.
  • PLGA with acid end groups was purchased from Adsorbable Polymers International.
  • a PEG polymer of molecular weight 3,400 with a terminal amine and carboxylic group (NH 2 -PEG-COOH) was purchased from JenKem
  • siRNAs targeting luciferase (siLuc), Revl (siREVl) and Rev3L (siREV3L) were synthesized by and purchased from Dharmacon. Cy3- labeled siRNA was purchased from IDT. The siRNA sequences are as follows:
  • siLuc sense 5'-CUUACGCUGAGUACUUCGA-3 '; (SEQ ID NO:3);
  • siREVl sense: 5'-GCUGUAUAAUGCAUGUUGAUA-3'; (SEQ ID NO:5); antisense: 5'-UAUCAACAUGCAUUAUACAGC-3'; (SEQ ID NO:6); siREV3L sense: 5'-UCUUCUUGCUGGUUUGGAAAG-3'; (SEQ ID NO:7); amtisense: 5'-CUUUCCAAACCAGCAAGAAGA-3'; (SEQ ID NO:8); Instruments
  • ESI-MS analyses were performed on an Agilent 1 100 series instrument. Atomic absorption spectroscopic measurements were taken on a Perkin-Elmer AAnalyst 300 spectrometer.
  • LipofectamineTM 2000, and Quant-iTTM RiboGreen® RNA quantitation reagent were purchased from Invitrogen (Carlsbad, CA).
  • Dual-GloTM Luciferase Assay System was purchased from Promega (Madison, WI).
  • a HeLa cell line stably expressing firefly and Renilla luciferase (Dual-Luc HeLa) was obtained from Alnylam
  • the cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen) with high glucose, 10 v/v% FBS, 500 ⁇ g/mL zeocin, and 0.5 ⁇ g/mL puromycin.
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS 10 v/v% FBS
  • zeocin 500 ⁇ g/mL zeocin
  • puromycin 0.5 ⁇ g/mL puromycin.
  • LNCaP and MDA-MB-231 cell lines both bioluminescent and non-bioluminescent
  • ATCC American Type Culture Collection
  • Cells lines were cultured in RPMI-1640 media (Invitrogen, Carlsbad, CA) supplemented with 10% FBS and 1% penicillin/streptomycin.
  • Cells and biological experiments were conducted at 37°C in 5% C02.
  • G0-C14 was synthesized by reacting 1,2-epoxytetradecane with generation 0 of ethylenediamine core-PAMAM dendrimer according to a known procedure.
  • a substoichiometric amount of 1,2-epoxytetradecane was added to PAMAM dendrimer at a molar ratio of 7: 1.
  • the mixture was reacted under vigorous stirring at 90 °C for 2 days.
  • the crude reaction mixture was separated by chromatography on silica with a gradient elution from CH2CI2 to 75:22:3 CH 2 Cl 2 /MeOH/NH 4 OH.
  • the purified product was characterized by J H NMR.
  • NPs The design and preparation of polymer/G0-C14 hybrid NPs are shown in Figures 1A-C.
  • G0-C14 cationic lipid- like molecules termed G0-C14 was synthesized through ring-opening of 1,2- epoxytetradecane by generation 0 of poly(amidoamine) (PAMAM) dendrimers ( Figure 1C).
  • PAMAM poly(amidoamine) dendrimers
  • NPs were prepared through self-assembly of PLGA- b-PEG and the resulting G0-C14 using a double emulsion-solvent evaporation method.
  • the generation 0 of PAMAM dendrimer was used for the cationic lipid preparation due to its minimal cytotoxicity compared to higher generations, while still providing sufficient positive charge to entrap the siRNA therapeutic agent. It has been shown that lipid-like materials, termed lipidoids, containing a 14 carbon tail are ideal for siRNA delivery; thus, 1,2-epoxytetradecane was selected for synthesis of G0-C14.
  • Decanoic anhydride (0.41 g, 1.26 mmol, 3.8 equiv) was added to a solution of cw.cw, tra «5-[Pt(NH 3 ) 2 Cl 2 (OH) 2 ] (0.1 1 g, 0.33 mmol) in 10 mL
  • Compound 1 was designed and synthesized as a Pt(IV) prodrug because its hydrophobicity allowed for encapsulation within PLGA-b-PEG NPs while being sufficiently soluble in organic solvents like DMSO and dichloromethane (DCM), which is required for Pt(IV)-encapsulated NP preparation. Additionally, Pt(IV) complexes can be reduced in the intracellular milieu to yield the cytotoxic Pt(II) species through a reductive elimination of axial ligands. The redox potential for the reduction of a Pt(TV) prodrug, which is an analog of 1, has been previously investigated at various pH conditions.
  • Pt(IV) prodrugs provide an attractive alternative to the existing portfolio of Pt(II) drugs.
  • Copolymer PLGA-b-PEG was synthesized by the amide coupling of
  • NPs encapsulated with a combination payload of siRNAs and compound 1 was formulated via double emulsion solvent evaporation technique.
  • copolymer PLGA-b-PEG and G0-C14 were co-dissolved in dichloromethane (DCM) with or without compound 1.
  • siRNAs was reconstituted in HyPureTM molecular biology grade water (Hyclone Laboratories, Inc., Logan, Utah).
  • the siRNA solution (0.4 mL) was added drop-wise into 1 mL PLGA-b-PEG and G0-C14 solution and emulsified by probe sonification to form the first emulsion.
  • the emulsified mixture was added into 3 mL aqueous solution containing 1% PVA, followed by probe sonification to form the double emulsion.
  • the final emulsion solution was poured into 15 mL water and stirred for 3 hours to allow the DCM solvent to evaporate and the particles to harden.
  • the remaining organic solvent and free molecules were removed by washing the particle solution three times using an Amicon Ultra-4 centrifugal filter (Millipore, Billerica, MA) with a molecular weight cutoff of 100 kDa.
  • the NP size and zeta potential were determined by using a ZetaPALS dynamic light-scattering detector (15 mW laser, incident beam of 676 nm; Brookhaven Instruments Corporation, Holtsville, NY).
  • the size of the NPs range from 180 nm to 220 nm with
  • NPs with a fixed G0-C14/siRNA weight ratio of 20 were used. The NPs showed compact and spherical morphology with a mean diameter of around 200 nm.
  • Cy3-labeled siRNA was first encapsulated into the NPs.
  • a suspension of NPs in phosphate buffered saline (PBS) was aliquotted (100 ⁇ ,) into several semipermeable minidialysis tubes (molecular mass cutoff 100 kDa; Pierce) and dialyzed against frequently renewed PBS (pH 7.4) at 37 °C with gentle stirring.
  • PBS phosphate buffered saline
  • An aliquot of the NP suspension was removed, and the platinum content was analyzed by AAS.
  • siRNA quantification a standard curve correlating fluorescence and Cy3 -siRNA concentration was used to determine the amount of siRNA encapsulated within the NPs.
  • the fluorescence intensity was measured by Synergy HT multi-mode microplate reader (ex/em 530/590 nm, BioTek Instruments Inc., Winooski, VT).
  • the release kinetics of the Pt(IV) prodrug 1 and siRNA from the NPs were measured.
  • the Pt(IV) compound 1 is homogeneously dispersed by encapsulation throughout the hydrophobic PLGA layer and is released through a diffusion-controlled process and polymer degradation .
  • Release studies were conducted by dialyzing NPs containing both compound 1 and siRNA against 2 L of frequently renewed PBS at pH 7.4 and 37 °C to mimic physiological conditions.
  • the amount of platinum released from the NPs was measured by atomic absorption spectroscopy (AAS).
  • NPfCompoinid 1 The cellular uptake of " NPfCompoinid 1) and free Compound 1 was studied using a LNCaP cell line.
  • LnCaP cells were seeded into 6-well plates (2.5 x 10 6 cells/well) and allowed to adhere overnight in growth medium.
  • NP(Compound J) and free Compound ⁇ were added to the growth media at a final platinum concentration of 0.1 mM.
  • the cells were washed with PBS buffer, followed by treatment with nitric acid for 3 hours.
  • the cell lysate was collected and the cellular platinum concentration was quantitatively determined by AAS.
  • the platinum content was normalized against protein concentration in the cell extracts, measured by a BCA protein kit. Data were reported as mean standard deviation for triplicate samples and the experiment was repeated twice.
  • NP compound 1
  • NPs shield compound 1 from unspecific interactions with albumin present in the cell growth medium, which are considered to be the main route for platinum binding in human blood plasma.
  • Dual-Luc HeLa cells were seeded into 96-well plates (15,000 cells/well) and allowed to attach in growth medium at 37 °C in a 5% CO2 incubator overnight. Cells were then transfected with naked siLuc, NPs encapsulating siLuc (NP(siLuc)), blank NP, and Lipofectamine 2000 (Lipo2000) complexed siLuc. Lipo2000 and siRNA were mixed to form lipoplex according to the manufacturer's protocol as a positive control. After 24 hours, the cells were washed with fresh medium and further incubated in the medium for one day. The expression of firefly and Renilla luciferase in HeLa cells was determined by Dual-GloTM Luciferase assay kits (Promega). The luminescence intensity was measured using microplate reader (Tecan). All of the in vitro transfection experiments were performed in quadruplicate.
  • REV1IREV3L siRNA transfection of LNCaP and MDA-MB-231 cells was conducted in 6-well plates (400,000 cells/well). The cells were allowed to adhere overnight in growth medium and then were transfected with NP(si ?EP7, siREV3L). 24 hours post-transfection, the cells were washed with fresh medium and the cellular levels oiREVI and REV3L mRNAs were assessed at three consecutive days using qRT-PCR. In brief, the total RNA from transfected cells was extracted using the RNeasy mini-kits (Qiagen, Germantown, MD) according to the manufacturer's protocol. qRT-PCR assays were performed using SYBR green on a BioRad thermal cycler. Primer sequences used in qPCR are: human housekeeping gene GAPDH (glyceraldehyde 3 -phosphate dehydrogenase):
  • GACAAGCTTCCCGTTCTCAG SEQ ID NO: 10; human REV1:
  • CTGGGATCCATCGCTGTAGT SEQ ID NO: 14
  • Relative gene expression values were determined by the AACT method using StepOne Software (Applied Biosystems). Data are presented as the fold difference in target gene expression normalized to GAPDH as the endogenous reference, and relative to the untreated control cells.
  • NPs containing various doses of anti-firefly luciferase siRNA (siLuc) were incubated with cells in the presence of growth media and the expression of both reporter proteins was measured one day post-transfection.
  • siRNA-mediated silencing reduction in firefly luciferase expression in the absence of Renilla reduction was considered to be the consequence of siRNA-mediated silencing. Renilla expression was monitored as an internal control for nanocarrier-associated toxicity.
  • the gene knockdown efficacy of NPs(siLuc) was determined by the comparison of detected protein expression levels in treated groups against the untreated control and expressed as relative firefly luciferase expression.
  • NP The performance of the NPs is plotted as a function of siRNA dose as shown in Figure 3 A.
  • NP that lacked siRNA produced no silencing effects while luciferase expression was significantly silenced with an increase of siLuc dose in NPs.
  • NP(siLuc) achieved greater than 95% luciferase knockdown, a more efficient silencing efficacy than the commercially available liposome-based lipoplex (Lipo2000-siRNA complex).
  • XTT 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-
  • Dual-Luc cells were seeded in quadruplicate in 96-well plates (15,000 cells/well). The cells were allowed to adhere overnight in growth medium and then treated with formulations containing escalating-dose of NP(siLuc). Cells in media containing 10% FBS with nothing added were used as controls. 24 hours after treatment, cell viability was measured using XTT assay (Roche Applied Science, Indianapolis, IN) following the manufacturer's protocol.
  • the LNCaP cells were first transfected with W(s REVl, si ' REV3L) for 24h and the medium was replaced with fresh medium thereafter.
  • the first formulation consists of ⁇ NP(siREVl, s REV3L) with compound 1 in solution form and the second contains compound 1 within the NP NVisiREVl , s REV3L, compound 1)].
  • Untransfected cells were seeded and treated with escalating-dose of compound 1 either in solution or NP form. Cell viability was then measured using XTT assay 24 hours post treatment.
  • Luciferase-expressing xenograft tumors were induced in the mammary fat pad of 8-week-old BALB/C nude mice (Charles River
  • tumor volume (V) length ⁇ width ⁇ widtlv2.
  • the NP solution was concentrated to 15 mg/mL with the total encapsulated siRNA concentration being - 150 ⁇ g/mL.
  • siRNA-containing NPs were directly injected into the tumor at an equivalent dose of - 0.4 mg/kg entrapped siRNA.
  • the mice were monitored using an IVIS Spectrum bioluminescent and fluorescent imaging system (Caliper Life Sciences). Tumor bioluminescence images were then taken daily for 3 days post-injection. The bioluminescence intensity was analyzed using Livinglmage acquisition and analysis software. All of the in vivo imaging experiments were performed in quintuplicate.
  • LNCaP cells were first retrovirally infected to stably express GFP protein.
  • Retroviral vector used is well known MSCV/LTRmiR30-SV40-GFP which is under control of the SV40 promoter.
  • MSCV vector was transfected into 293T cell line by Calcium method to produce retro virus.
  • LNCaP cells were sorted on MoFlo flow cytometer.
  • the GFP-labeled LNCaP cells (10 6 cells, 0.1 mL) were injected subcutaneous ly into the flank of a male SCID-beige mouse using 50%> matrigel. Tumor nodules were allowed to grow to a volume of approximately 100 mm 3 prior to treatment.
  • the mouse LNCaP xenograft tumor model was developed by injecting 0.1 mL LNCaP cell suspension (10 6 cells) into the flank of a male SCID-beige mouse using 50% matrigel. Tumor nodules were allowed to grow to a volume of approximately 100 mm 3 before initiating treatment. Tumor-bearing mice were randomly assigned to five groups prior to treatment, and their weight and the initial tumor volume were measured and recorded, respectively. Starting from day 4, test animals received two intratumoral injections weekly at intervals of 3 or 4 d of (i) saline, (ii) compound 1 in solution form, (iii) compound 1 encapsulated NP
  • mice Prior to treatment with the two siRNA-containing NP formulations (regimens (iv) and (v)), the mice were injected with NP(siftEF/, siREV3L) on day 0 and day 2 at an equivalent dose of - 0.4 mg/kg entrapped siRNA in order for REV1IREV3L depletion to occur at noticeable levels.
  • injections were initiated with the aforementioned 5 formulations at an equivalent dose of 4 mg/kg for compound 1 and 0.4 mg/kg for siRNA per injection.
  • the weight and tumor volume of each mouse were measured twice weekly over a period of 37 days.
  • cancer drugs including platinum-based compounds and cyclophosphamide
  • attack cancer cells by damaging their DNA This DNA damage can prevent cells from replicating their DNA before dividing, which usually induces apoptosis.
  • cancer cells can use enzymes known as translesion DNA polymerases to carry out TLS, allowing them to escape apoptosis.
  • This type of DNA replication is highly prone to errors, thereby introducing mutations into the DNA.
  • These newly acquired mutations can allow cancer cells that survive chemotherapy to be much more drug-resistant and aggressive.
  • error-prone TLS can induce massive genomic mutations after DNA-damaging chemotherapy while also helping the cells avoid the cytotoxic effect of the treatment.
  • Revl is a scaffolding protein that recruits other translesion DNA polymerases to DNA lesions. It is also a dCMP transferase that contributes to the bypass of certain lesions.
  • Rev3L is the catalytic subunit of DNA polymerase ⁇ (Pol ⁇ ), which plays a key role in extending replication termini across DNA damage. It has been found that human cells expressing reduced levels of genes REV1 and REV3L are more sensitive to cytotoxicity by cisplatin. Thus, it was hypothesized that the suppression of REV 1 and REV3L by specific siRNA-containing NPs would inhibit TLS activity, impair drug-induced mutagenesis, and consequently sensitize cancer cells to chemotherapy.
  • NP(siREVl, siREV3L) was determined. After 24 hours incubation with NPs, the cells were washed with fresh medium and the cellular levels of REVl and REV3L mRNA were assessed on three consecutive days using quantitative real-time PCR (qRT-PCR).
  • the qRT-PCR assay revealed a sustained knockdown efficiency of up to 87% for both genes REVl and REV3L in LNCaP cells over the course of three days.
  • a similar gene silencing efficacy was observed in MDA-MB-231 cells ( Figure 6).
  • these developed NPs are capable of simultaneously targeting multiple genes.
  • An escalating- dose experiment in LNCaP cells was done to examine the effects of NP- mediated REV1/REV3L suppression on tumor cell chemosensitivity.
  • the following four different formulations were compared: (i) compound 1 in solution form, (ii) compound 1 encapsulated NP [NP(compound 1)], (iii) NP(siREVl, siREV3L) with compound 1 in solution, and (iv) NP(siREVl, siREV3L, compound 1).
  • the cells Prior to treatment with the two siRNA-containing NP formulations, the cells were transfected with NP(siREVl, siREV3L) for 24 hours in order to achieve substantial levels of Rev 1 and Rev3L suppression. The transfected cells were then treated with the two different siRNA-containing formulations with escalating-dose of compound 1.
  • the first formulation consists of NP(siREVl, siREV3L) with compound 1 in solution form, whereas the second contains compound 1 within the NP
  • NP(siREVl, siREV3L, compound 1) A comparison of the dose-response curves revealed significantly lower EC50 values for all three NP formulations when compared to free drug in solution form (Figure 5B). As expected, the cells treated with NP(siREVl, siREV3L) exhibited better dose- response to free drug than the untreated cells, thus providing evidence for the enhanced chemosensitization of prostate cancer cells through REV1 and REV3L suppression. Similarly, inclusion of the EV1/ EV3L siRNAs in NPs resulted in improved tumor cell response to compound 1 -loaded NPs compared to NP(scrambled siRNA, compound 1) ( Figure 7).
  • LNCaP low-density polypeptide
  • GFP green fluorescent protein
  • NP(siREVl, siREV3L) (experimental group) at a dose of 0.4 mg siRNA per kg animal via intratumoral injection.
  • Tumors were harvested from the mice at the designated time points and pure populations of LNCaP cells were isolated by GFP sorting before qRT-PCR.
  • mice were randomly divided into 5 groups and treated with the following regimens with equivalent doses of compound 1 via intratumoral administration twice weekly for 5 weeks: (i) saline, (ii) compound 1 in solution form, (iii) compound 1 encapsulated NP [NP(compound 1)], (iv) NP(siREVl, siREV3L) with compound 1 in solution, and (v) NP(siREVl, siREV3L, compound 1).
  • mice prior to treatment with the two siRNA-containing NP formulations [regimens (iv) and (v)], mice were injected on day 0 and day 2 with NP(siREVl, siREV3L) to ensure that Revl and Rev3L depletion occurred at significant levels.
  • injections with the aforementioned 5 formulations were initiated.
  • the aim of this study was to determine whether simultaneous delivery of REV1/REV3L specific siRNAs and platinum chemotherapeutics would result in enhanced antitumor activity through synergistic effects.
  • Figure 8A showed that the cellular levels of REV 1 and REV3L expression were significantly decreased by 78% relative to the control in the harvested GFP-labeled LNCaP cells 48 hours post-treatment. Additionally, the gene silencing effect mediated by a single dose of NP(siREVl, siREV3L) was still noticeable 72 hours after administration (36% and 42% decrease in REV1 and REV3L, respectively). In this context, these results further demonstrate that the NPs are able to efficiently deplete REVl and REV3L expression in tumors.
  • NP(scrambled siRNA, compound 1) group further emphasizing that suppressing REV1/REV3L expression can enhance tumor growth suppression (Figure 8B and 9A). These results remain consistent with the in vitro escalating-dose experiment ( Figure 5B and 7). Strikingly, all mice treated with NP(siREVl, siREV3L, compound 1) survived the entire 50-day study duration without tumor growth ( Figure 8C). In contrast, no other group had animals survive the 34-day study ( Figure 8C and 9B). The combination of NP(siREVl, siREV3L) with compound 1 in solution (regimen (iv)) was also more efficacious with respect to survival than the free drug,
  • a nanoparticle comprising
  • a shell comprising one or more hydrophobic cationic moieties, one or more amphiphilic moieties comprising a biodegradable polymer and a hydrophilic polymer, and one or more hydrophobic therapeutic, prophylactic, and/or diagnostic agents.
  • nanoparticle of claim 1 wherein the nucleic acid is siRNA.
  • hydrophobic cationic moiety comprises a cationic small molecule, polymer, or dendrimer.
  • hydrophobic cationic moiety comprises a hydrophobic group selected from the group consisting of lipophilic alkyl groups, cholesterol, or combinations thereof.
  • nanoparticles of claim 8 wherein the agent is homogeneously dispersed in the shell.
  • platinum-based chemotherapeutic agent is cisplatin, derivative thereof, or a prodrug thereof.
  • a method of making the particle of any one of claims 1-15 comprising dissolving the biodegradable polymeric material and the amphiphilic compound in a water immiscible organic solvent; separately dissolving an agent in an aqueous solution, optionally containing one or more water miscible solvents; and adding the aqueous solution to the water immiscible organic solution to form an emulsion.

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Abstract

La présente invention concerne des nanoparticules contenant un noyau aqueux contenant un ou plusieurs acides nucléiques, tels que des ARNsi, et une enveloppe contenant un ou plusieurs matériaux cationiques hydrophobes, un ou plusieurs matériaux amphiphiles, et un ou plusieurs agents thérapeutiques, diagnostiques et/ou prophylactiques. Le matériau cationique hydrophobe et la partie hydrophobe du matériau amphiphile constituent une matrice polymère non polaire pour charger des médicaments non polaires, protéger et favoriser la rétention de molécules d'ARNsi à l'intérieur du noyau NP, et contrôler la libération de médicament. La partie hydrophile du matériau amphiphile peut former une couronne autour de la particule qui prolonge la circulation des particules dans la circulation sanguine et diminue l'absorption par le RES.
PCT/US2014/061185 2013-10-17 2014-10-17 Nanoparticules cationiques pour co-administration d'acides nucléiques et d'agents thérapeutiques WO2015058111A1 (fr)

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